文章摘要
人工模拟条件下环境DNA宏条形码技术的定量分析初探
The linear relationship for quantitative analysis by eDNA Metabarcoding under ideal conditions
投稿时间:2020-05-19  修订日期:2020-05-22
DOI:
中文关键词: 环境DNA宏条形码,PCR引物偏好,高通量测序,线性函数关系,生物学调查
英文关键词: eDNA Metabarcoding, primer bias, high-throughput sequencing, linear relationship, biological investigation
基金项目:山东省支持青岛海洋科学与技术试点国家实验室重大科技专项
作者单位E-mail
牟铭 黄海水产研究所 1065028922@163.com 
李昂 黄海水产研究所  
赵新宁 黄海水产研究所  
柳淑芳 黄海水产研究所 liusf@ysfri.ac.cn 
庄志猛 黄海水产研究所  
摘要点击次数: 80
全文下载次数: 0
中文摘要:
      近年来,环境DNA宏条形码技术(eDNA Metabarcoding)在水生生态系统的物种检测、生物多样性评估等领域中得到广泛应用,因其具有快速测算群落中物种丰度的潜能,eDNA宏条形码技术成为资源保护和管理中颇具有应用前景的调查工具。虽然大量证据表明eDNA高通量测序获得的reads数与自然环境中生物相对数量具有相关性,但一直不能得出明确的量化关系结果。主要原因是,从环境生物到实验室检测数据,eDNA的富集效率难以评估,同时eDNA扩增过程中不可避免地出现引物偏倚现象,导致了eDNA宏条形码技术的检测结果存在诸多不确定性,从而制约了该技术在生物资源调查领域的推广应用。假定水体中的eDNA全部回收,且PCR扩增时不存在引物偏倚性,这种理想状态下的水体中eDNA组成与其高通量测序reads数是否存在线性关系?为此,本研究在实验室可控条件下,选择凡纳滨对虾和墨吉对虾两个同属近缘种,对其DNA样品进行不同比例混合,模拟从自然水体中富集到的eDNA复合样品,既保证了样品的回收率,又降低了引物偏倚的干扰。以此为模板,探究eDNA宏条形码技术检测种群相对数量的准确性。结果发现当两个物种DNA模板浓度比例为1:1时,高通量测序结果注释得到的两个物种reads数比值为13/24(墨吉对虾/凡纳滨对虾),可见,即使是同属近缘种间依然存在轻微的引物偏倚现象,引物偏移率为1.5%。同时,根据七个试验组获得的高通量测序结果注释得到的两个物种reads数比值与对应模板中两个物种DNA浓度比值之间的线性回归分析表明,水体中eDNA组成与其高通量测序reads数间呈明显线性关系,即y = 0.0716x + 0.7043(r2 = 0.9824)。综上,本研究为验证eDNA宏条形码技术监测水生生物资源量的可行性提供了直接证据,也为后续DNA宏条形码技术的定量研究提供了思路。
英文摘要:
      In recent years, eDNA metabarcoding has been widely used in the fields of biological investigation, which includes species detection, biodiversity assessment, etc. Because eDNA Metabarcoding has the potentiality to rapidly assess species abundance in communities, it has become a promising investigation tool in resources conservation and management. Although there were lots of reports about eDNA Metabarcoding, which indicated that the high-throughput sequencing (HTS) reads were related to the biomass in the natural environmental sample, we could not get a clear quantitative relationship between them.. From field to the laboratory, the enrichment efficiency of eDNA is difficult to evaluate. Meanwhile, primer bias inevitably occurs in the eDNA amplification process, resulting in the uncertainty of eDNA HTS results, which restricts the application of eDNA metabarcoding in the field of biological resource investigation. Assuming eDNA to be recovered completely, and no primer bias during PCR amplification, that in this ideal state, whether there is a linear relationship between eDNA and HTS reads? In this study, under controlled conditions in the laboratory, two closest relatives, Penaeus vanamei and Penaeus merguiensis, were selected, and their DNA samples were mixed in different proportions to simulate eDNA samples which enriched from natural waters. In this way, the recovery of the sample is the highest and the primer bias is the lowest.. Using this eDNA template to explore the accuracy of eDNA metabarcoding in detecting species biomass. The results showed that when the concentration ratio of two species DNA template was 1:1, the HTS reads number ratio of two species is 13/24 (Penaeus merguiensis / Penaeus vanamei). So there is a slight primers bias even between the closest relatives (primers migration rate was 1.5%). At the same time, according to the HTS results obtained from the seven test groups, it showed an obvious linear relationship between the composition of eDNA in the water and the number of high-throughput sequencing reads, that is, y = 0.0716x + 0.7043 (r = 0.9824). In summary,, this study provides direct evidence to verify the feasibility of eDNA metabarcoding in monitoring the aquatic biological resources, and also provides ideas for the subsequent quantitative study of DNA metabarcoding.
附件
View Fulltext   查看/发表评论  下载PDF阅读器
关闭